This invention relates generally to the nematode parasite Dirofilaria immitis and, more particularly, to newly characterized circulating parasite antigens present in the blood of animals infected with the nematode parasite Dirofilaria immitis, to novel hybrid cell lines that produce monoclonal antibodies to such parasite antigens, to the monoclonal antibodies themselves, and to diagnostic methods and compositions employing such antibodies in the detection of such previously uncharacterized specific parasite antigens as a means of diagnosing and quantifying parasitic infections.
Dirofilaria immitis (the common dog heartworm) is a filarial nematode parasite that is an important pathogen of canines in the United States and many other countries. Although the parasite can cause heart failure, lung disease and death, infected animals often have no outward evidence of disease. Signs of heartworm disease in dogs, when present, are nonspecific. The diagnosis of D. immitis infection is most commonly made by demonstrating microfilariae, larval forms of the parasite, in peripheral blood smears. This time-honored test is insensitive. It is now well recognized that a significant proportion of infected dogs lack microfilaremia. Microfilarial examination also suffers from a lack of specificity. Animals infected with nonpathogenic filarial parasites have circulating microfilariae that are difficult to distinguish from those of D. immitis. A number of serologic tests have been described that measure serum antibodies specific for parasite antigens as a means of diagnosing D. immitis infections. Unfortunately, these tests lack specificity. Uninfected dogs often have antibodies that react with D. immitis antigens because of prior infection, infection with related parasites, or exposure to the parasite without the development of mature infection.
A further difficulty with existing diagnostic techniques for D. immitis infection is that neither microfilarial counts nor antibody titers correlate significantly with the intensity of infection, the number of adult worms in the animal. Infection intensity may relate to the prognosis of infection in dogs with or without therapy. In summary, currently available parasitological and serological tests cannot reliably diagnose or quantitate D. immitis infections.
Difficulties with existing diagnostic techniques have inspired efforts to develop assays for parasite antigens that would have the potential of being sensitive, specific and related to infection intensity. The desire for antigen detection in parasitology is part of a general trend in microbiology toward assays that detect microbial antigen as a substitute for cultivation of the agent or measurement of antibody. Such assays have been particularly useful for pathogens that cannot be grown in vitro (i.e., Hepatitis B, rotovirus, Pneumocystis) or where rapid diagnosis of a serious illness can influence therapy before culture results are available (i.e., Cryptococcus, Hemophilus, or pneumococcal meningitis).
The first report of circulating parasite antigens in filariasis was published in 1946 by Franks (Journal of Parasitology, 32:400) who found that serum from filariasis patients caused a wheal and flare reaction when it was injected into skin that was passively sensitized with serum containing antifilarial antibodies. Thirty years later, Desowitz and Una (Journal of Helminthology, 50:53, 1976), who were using counterimmunoelectrophoresis (CIE) to measure antibody to D. immitis in dogs, reported the incidental finding that 2 of 5 infected dogs had circulating parasite antigen in serum. CIE has also been used to detect circulating parasite antigens in rats infected with filarial worms and in limited studies with human sera (e.g., see Dasgupta and Bala, Indian Journal of Medical Research, 67:30, 1978; and Kaliraj et al, Journal of Helminthology, 55:133, 1981). Until recently, however, no attempt had been made to identify the specific antigens that circulate in filarial infections or to develop more sensitive assays to detect such antigens.
In 1980, Ouassi et al. (American Journal of Tropical Medicine and Hygiene, 30:1211) developed a test for circulating onchocerciasis antigens based on polyvalent rabbit antiserum which detected antigenemia in 75% of infected patient sera. The major antigen detected was cathodic and trichloroacetic acid soluble. The same group more recently developed a monoclonal antibody specific for the circulating onchocerciasis antigen and they have successfully used the antibody to detect antigen in patient sera (Des Moutis et al., American Journal of Tropical Medicine and Hygiene, 32:533, 1983).
My own group (Weil et al., American Journal of Tropical Medicine and Hygiene, 33:425 (1984)) has focused on the canine-Dirofilaria immitis system, using counterimmunoelectrophoresis modified to increase sensitivity and detect antigens within immune complexes. Circulating antigens were detected in 24 of 24 infected dog sera independent of microfilarial patency. No false positive tests were observed in sera from 26 control dogs that were free of infection at necropsy or in sera from 13 dogs infected with the filarial worm Dipetalonema reconditum.
Hamilton et al. (Federation Proceedings, 43:854, 1983) have reported on efforts to detect circulating D. immitis antigens in dog sera with polyclonal (rabbit) antibody-based radioimmunoassays. They found that their ability to detect parasite antigens (unspecified and uncharacterized) was decreased in serum from dogs that contained high levels of specific antibody.
Scott et al. (Federation Proceedings, 42:1089, 1983) have reported the production of monoclonal antibodies to Dirofilaria immitis antigens obtained by fusing mouse myeloma cells with spleeh cells of mice that had been immunized with D. immitis adult and microfilarial antigens. Two of the monoclonal antibodies were used together in an enzyme-linked immunosorbent assay (ELISA) to detect D. immitis antigens that had been added to normal dog sera with a sensitivity of 25 ng/ml. No description of the actual parasite antigens present within infected dog serum was provided. Similarly, there was no description of the detection of parasitic antigens in infected dog sera.
In the above-noted studies by Des Moutis et al. and Scott et al., monoclonal antibodies were produced by immunizing with crude antigen extracts and screening for antibody production either with TCA-extracted antigen (Des Moutis et al.) or the crude antigen extract (Scott et al.). To date there have been no reports on the nature of the parasite antigens that circulate in the serum of D. immitis-infected dogs. Accordingly, these antigens have not been characterized nor have monoclonal antibodies specific for such antigens been prepared as a means of diagnosing and quantifying parasitic infections caused by D. immitis.